Crystal structure of a bifunctional transformylase and cyclohydrolase enzyme in purine biosynthesis.

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.

Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity.

The bifunctional enzyme aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) is responsible for catalysis of the last two steps in the de novo purine pathway. Gel filtration studies performed on human enzyme suggested that this enzyme is monomeric in solution. However, cross-linking studies performed on both yeast and avian ATIC indicated that this enzyme might be dimeric. To determine the oligomeric state of this protein in solution, we carried out sedimentation equilibrium analysis of ATIC over a broad concentration range. We find that ATIC participates in a monomer/dimer equilibrium with a dissociation constant of 240 +/- 50 nM at 4 degrees C. To determine whether the presence of substrates affects the monomer/dimer equilibrium, further ultracentrifugation studies were performed. These showed that the equilibrium is only significantly shifted in the presence of both AICAR and a folate analog, resulting in a 10-fold reduction in the dissociation constant. The enzyme concentration dependence on each of the catalytic activities was studied in steady state kinetic experiments. These indicated that the transformylase activity requires dimerization whereas the cyclohydrolase activity only slightly prefers the dimeric form over the monomeric form.

Crystallization and preliminary crystallographic investigations of avian 5-aminoimidazole-4-carboxamide ribonucleotide transformylase-inosine monophosphate cyclohydrolase expressed in Escherichia coli.

ATIC [5-aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase)-inosine monophosphate cyclohydrolase (IMPCH)] is a bifunctional enzyme that catalyzes the penultimate and final steps in the de novo purine biosynthesis pathway and thus is an attractive anticancer target. Recombinant avian ATIC has been purified from an Escherichia coli expression system and crystallized in a binary complex with methotrexate (MTX). Crystals were obtained from PEG 4000 or MPEG 5000 buffered at pH 7.0-7.2 and data were collected from a single crystal at 96 K to 2.3 A resolution at the Stanford Synchrotron Radiation Laboratory (SSRL). The crystals are monoclinic and belong to space group P2(1), with unit-cell dimensions a = 65.17, b = 105.93, c = 103.47 A, beta = 108.27 degrees. Assuming two molecules per asymmetric unit, the Matthews coefficient V(m) is 2.63 A(3) Da(-1) and the solvent volume is 52.9%.

Dihydrofolate reductase from Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the first human virus known to encode dihydrofolate reductase (DHFR), an enzyme required for nucleotide and methionine biosynthesis. We have studied the purified KSHV-DHFR enzyme in vitro and analyzed its expression in cultured B-cell lines derived from primary effusion lymphoma (PEL), an AIDS-associated malignancy. The amino acid sequence of KSHV-DHFR is most similar to human DHFR (hDHFR), but the viral enzyme contains an additional 23 amino acids at the carboxyl-terminus. The viral DHFR, overexpressed and purified from E. coli, was catalytically active in vitro. The K(m) of KSHV-DHFR for dihydrofolate (FH(2)) was 2.4 microM, which is significantly higher than the K(m) of recombinant hDHFR (rhDHFR) for FH(2) (390 nM). K(m) values for NADPH were similar for the two enzymes, about 1 microM. KSHV-DHFR was inhibited by folate antagonists such as methotrexate (K(i): 200 pM), aminopterin (K(i): 610 pM), pyrimethamine (K(i): 29 nM), trimethoprim (K(i): 2.3 microM), and piritrexim (K(i): 3.9 nM). In all cases, K(i) values for these folate antagonists were higher for KSHV-DHFR than for rhDHFR. The viral enzyme was expressed at levels two- to tenfold higher than hDHFR in PEL cell lines as an early lytic cycle gene. KSHV-DHFR mRNA and protein appeared from 6 to 24 h after chemical induction of the KSHV lytic cycle. Epitope-tagged KSHV-DHFR and rhDHFR both localized to the nucleus of transfected cells, while other KSHV nucleotide metabolism genes localized to the cytoplasm. DHFR activity was not essential for viral replication in cultured PEL cells. Since hDHFR was not detectable in peripheral blood mononuclear cells (PBMCs), KSHV-DHFR may function to provide increased DHFR activity in vivo in infected cells that have little or none of their own enzyme.

Expression of HER2/erbB-2 correlates with survival in osteosarcoma.

PURPOSE: In osteosarcoma, prognostic factors at diagnosis other than clinical stage have not been clearly identified. The aim of this study was to determine whether human epidermal growth factor receptor 2 (HER2)/erbB-2, p-glycoprotein, or p53 expression correlated with histologic response to preoperative chemotherapy or event-free survival. PATIENTS AND METHODS: We performed a retrospective immunohistochemical study on material obtained from patients treated on the Memorial Sloan-Kettering Cancer Center T12 protocol between 1986 and 1993. Paraffin-embedded tissue was identified from 53 patients (73% of patients enrolled onto protocol) and stained for HER2/erbB-2, p53, and p-glycoprotein expression using standard monoclonal antibodies and methods. RESULTS: At the time of initial biopsy, 20 (42.6%) of 47 samples demonstrated high levels of HER2/erbB-2 expression. Higher frequencies of expression were observed in samples from patients with metastatic disease at presentation and at the time of relapse. Expression of HER2/erbB-2 correlated with a significantly worse histologic response (P =.03). In patients presenting with nonmetastatic disease, expression of HER2/erbB-2 at the time of initial biopsy was associated with a significantly decreased event-free survival (47% v 79% at 5 years, P =.05). p53 and p-glycoprotein expression did not correlate with histologic response or patient event-free survival. CONCLUSION: The correlation of HER2/erbB-2 expression with histologic response to preoperative chemotherapy and event-free survival in this study suggests that HER2/erbB-2 should be evaluated prospectively as a prognostic indicator. The correlation also suggests that clinical trials of antibodies that target this receptor, such as recombinant humanized anti-HER2 monoclonal antibody (Herceptin; Genentech, San Francisco, CA), should be considered for the treatment of osteosarcoma.

Structure and functional relationships in human pur H.

1. The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced. The gene product, hPurH has been overexpressed in E. coli, purified to homogeneity and crystallized. 2. The human pur H gene lies on chromosome 2, between band q34 and q35. There is at least one intron of 278 bp near the 5' end. 3. Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5. The existence of a linker or interaction region between the catalytic domains remains to be established. 4. Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first. 5. The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2. 6. Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting. 7. A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits. Probable binding sites for AICAR and FAICAR can be identified on each monomer. 8. Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM. 9. The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding. Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266. These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.

Accumulation of methotrexate polyglutamates, ploidy and trisomies of both chromosomes 4 and 10 in lymphoblasts from children with B-progenitor cell acute lymphoblastic leukemia: a Pediatric Oncology Group Study.

Levels of accumulation of methotrexate polyglutamates were measured in vitro in lymphoblasts obtained at diagnosis from children with B-progenitor cell acute lymphoblastic leukemia (pro-B ALL). They were compared to numerical and structural chromosomal abnormalities present in these leukemic cells. In a series of 95 patients, the percent with high lymphoblast methotrexate polyglutamate levels increased with the increase in modal number of total chromosomes (p<0.001). Thus, lymphoblast methotrexate polyglutamate accumulation appeared to be closely linked to the extent of hyperdiploidy in childhood pro-B ALL. Lymphoblasts from 35 (88%) of the 40 children with hyperdiploid (>50 chromosomes) and 23 (88%) of 26 with hyperdiploid (DNA Index >1.16) pro-B ALL accumulated high levels of methotrexate polyglutamate, suggesting that they were more sensitive to methotrexate cytotoxicity. While children with hyperdiploid (DNA Index >1.16) pro-B ALL have a good prognosis, those with trisomies of both chromosomes 4 and 10, almost all of whom are hyperdiploid, have an even better outcome. There was no significant difference in methotrexate polyglutamate levels in lymphoblasts from 19 children with and 21 without trisomies of both chromosomes 4 and 10 (p = 0.25). The improved response to multi-agent chemotherapy conferred by the presence of trisomies of both chromosomes 4 and 10 in such patients may be due to increased sensitivity of their lymphoblasts to one or more anti-leukemic agents in addition to methotrexate.

Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

The nucleoside analog 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, DHPG) is an antiviral drug that is used in the treatment of a variety of herpes viruses in immunocompromised patients and in a gene therapy protocol that has shown promising activity for the treatment of cancer. To probe the structural effects of ganciclovir when incorporated into DNA, we determined and compared the solution structure of a modified ganciclovir-containing decamer duplex [d(CTG)(ganciclovir)d(ATCCAG)]2 and a control duplex d[(CTGGATCCAG)]2 using nuclear magnetic resonance techniques. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H and 31P NMR experiments. Proton-proton distances determined from NOESY data and dihedral angles determined from DQF-COSY data were used in restrained molecular dynamics simulations starting from canonical A- and B-form DNA models. Both the control and ganciclovir sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures that were in excellent agreement with the observed NOE intensities. Examination of the final ganciclovir-containing structures reveals that the base of the ganciclovir residue is hydrogen bonded to its complementary dC and is stacked in the helix; in fact, the base of ganciclovir exhibits increased stacking with the 5' base relative to the control. Interestingly, some of the most significant distortions in the structures occur 3' to the lesion site, including a noticeable kink in the sugar-phosphate backbone at this position. Further examination reveals that the backbone conformation, sugar pucker, and glycosidic torsion angle of the residue 3' to the lesion site all indicate an A-type conformation at this position. A possible correlation of these structural findings with results obtained from earlier biochemical studies will be discussed.

LY231514, a pyrrolo[2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes.

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.

The human purH gene product, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase. Cloning, sequencing, expression, purification, kinetic analysis, and domain mapping.

We report here the cloning and sequencing of the cDNA, purification, steady state kinetic analysis, and truncation mapping studies of the human 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase). These steps of de novo purine biosynthesis, respectively. In all species of both prokaryotes and eukaryotes studied, these two activities are present on a single bifunctional polypeptide encoded on the purH gene. The human purH cDNA is 1776 base pairs in length encoding for a 591-amino acid polypeptic (Mr = 64,425). The human and avian purH cDNAs are 75 and 81% similar on the nucleotide and amino acid sequence level, respectively. The Km values for AICAR and (6R,6S)10-formyltetrahydrofolate are 16.8 microM +/- 1.5 and 60.2 microM +/- 5.0, respectively, for the cloned, purified human enzyme. A 10-amino acid sequence within the COOH-terminal portion of human AICARFT/IMPCHase has some degree of homology to a previously noted "folate binding site." Site directed mutagenesis studies indicate that this sequence plays no role in enzymatic activity. We have constructed truncation mutants which demonstrate that each of the two enzyme activities can be expressed independent of the other. IMPCHase and AICARFT activities are located within the NH2-terminal 223 and COOH-terminal 406 amino acids, respectively. The truncation mutant possessing AICARFT activity displays steady state kinetic parameters identical to those of the holoenzyme.

Chiral chemical synthesis of DNA containing (S)-9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and effects on thermal stability, duplex structure, and thermodynamics of duplex formation.

The antiviral compound 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG, Ganciclovir) is used clinically to treat cytomegaloviral infections in immunocompromised hosts and more recently is being investigated as a chemotherapeutic agent to be used in combination with retroviral gene therapy. Structurally, DHPG, an analog of guanosine, lacks the 2'-deoxyribose carbon atom and is acyclic. It is therefore prochiral at the 4'-deoxyribose carbon, having both pro-R and pro-S isomers. This stereochemistry is critical during biochemical conversions. DHPG retains the equivalent of 3'-hydroxyl and 5'-hydroxyl moieties. These can potentially support not only initial misincorporation of DHPG into DNA but also subsequent nucleotide addition. The mechanism of DHPG antiviral action may thus not strictly be through chain terminations. To investigate the structural and biochemical consequences of incorporation of DHPG into DNA, with particular attention to the relative contributions of the deoxyribose sugar to the overall structure and stability of DNA, we have developed a methodology for the chiral chemical synthesis of DNA oligomers containing DHPG. The stereochemistry of the DHPG phosphoramidite was established by a stereoselective acetyl transfer reaction catalyzed by porcine pancreatic lipase. The DNA resisted enzymatic digestion at DHPG sites. Circular dichroism and copper phenanthroline cleavage studies indicated that the incorporation of DHPG into DNA does not significantly perturb the global B-conformation structure. Detailed thermodynamic investigations into DNA containing DHPG revealed reduced thermal stability, as evidenced by a decrease in melting temperature, with significant alteration of the enthalpy, entropy, and free energy of duplex formation. These data demonstrate that an intact deoxyribose ring significantly contributes to the stability of a DNA duplex.

Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM.

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.

Solution structure of a DNA dodecamer containing the anti-neoplastic agent arabinosylcytosine: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement.

The effect of araC incorporation into the dodecamer duplex [d(CGCGAATT) (araC)d(GCG)]2 was examined by comparing its nuclear magnetic resonance (NMR)-determined solution structure with that of the control duplex d[(CGCGAATTCGCG)]2. 1H and 31P resonances in both duplexes were assigned using a combination of 2-D 1H NMR and a 3-D 31P-1H heteroTOCSY-NOESY experiment. Proton-proton distances (determined from NOESY data) and sugar dihedral angles (from NOESY and COSY data) were used in restrained molecular dynamics simulations starting from canonical A- or B-form DNA models. Both the control and araC sets of simulations converged to B-type structures. These structures were subjected to full relaxation matrix refinement to produce final structures which were in excellent agreement (R1/6 < 0.05) with the observed NOE intensities. A detailed comparison of the final control and araC structures revealed a global similarity (overall RMSD approximately 1.3 A), with significant differences localized at the araC site and neighboring bases. These included changes in sugar pucker, backbone torsion angles, base stacking, and other helical parameters. These findings are in general agreement with the previously published X-ray structure of a decamer duplex containing araC. One intriguing feature of the NMR solution structure not found in the crystal structure is the presence of an intramolecular hydrogen bond between the 2' hydroxyl on the araC sugar and the 3' phosphate group.

Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase.

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.

Novel pyrrolo[2,3-d]pyrimidine antifolate TNP-351: cytotoxic effect on methotrexate-resistant CCRF-CEM cells and inhibition of transformylases of de novo purine biosynthesis.

N-[4-[3-(2,4-Diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (TNP-351), characterized by a pyrrolo[2,3-d]pyrimidine ring, is a novel antifolate that exhibits potent antitumor activities against mammalian solid tumors. The mechanism of action of TNP-351 was evaluated using some methotrexate-resistant CCRF-CEM human lymphoblastic leukemia cell lines as well as partially purified enzymes folylpolyglutamate synthetase (FPGS), aminoimidazolecarboxamide ribonucleotide transformylase (AICARTFase), and glycinamide ribonucleotide transformylase (GARTFase) from parent CCRF-CEM cells. TNP-351 was found to inhibit the growth of L1210 and CCRF-CEM cells in culture, with the doses effective against 50% of the cells (ED50 values) being 0.79 and 2.7 nM, respectively. The growth inhibition caused by TNF-351 was reversed by leucovorin or a combination of hypoxanthine and thymidine. The methotrexate-resistant CCRF-CEM cell line, which has an impaired methotrexate transport, showed less resistance to TNP-351 than to methotrexate. TNP-351 was also found to be an excellent substrate for FPGS with a Michaelis constant (Km) of 1.45 microM and a maximum of velocity (Vmax) of 1,925 pmol h-1 mg-1. Inhibitory activities of TNF-351-Gn (n = 1-6) for AICARTFase were found to be significantly enhanced with increasing glutamyl chain length [inhibition constants (Ki): G1, 52 microM; G6, 0.07 microM]. Neither TNP-351 nor its polyglutamates were very strong inhibitors of GARTFase. These findings have significant implications regarding the mechanism of action of TNP-351.

5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines.

5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly converted to high chain length polyglutamates. Transport via this system was inhibited in folate-depleted cells by 10 nM folic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a relationship between intracellular GTP levels and the induction of HL-60 leukemia cell differentiation by 5,10-dideazatetrahydrofolic acid (DDATHF).

5,10-Dideazatetrahydrofolic acid (DDATHF) is an inhibitor of glycinamide ribonucleotide transformylase, the first of two tetrahydrofolate requiring enzymes in the de novo purine nucleotide biosynthetic pathway, and is a potent inducer of the maturation of HL-60 promyelocytic leukemia cells. The inhibition of cellular growth by DDATHF was effectively prevented by adenosine or deoxyadenosine, whereas guanosine or deoxyguanosine only partially prevented the growth inhibition produced by this folate antimetabolite, implying that the depletion of both ATP and GTP, which occurs with this agent, was responsible for its growth inhibitory effects. In contrast, the induction of differentiation by DDATHF was completely abolished by the presence of guanosine or deoxyguanosine, suggesting that the depletion of intracellular guanine nucleotides by DDATHF represents the event that is essential to the induction of differentiation by this folate analog. This possibility was supported by the observation that the concentration of dGTP was not decreased in cells treated with DDATHF under the conditions employed. Both guanine nucleosides selectively restored intracellular GTP pools depleted by the treatment with DDATHF to their normal level, whereas only adenine nucleosides completely restored the levels of both ATP and GTP to their normal intracellular concentrations. The relationship between guanine nucleotide pools and the induction of HL-60 differentiation by DDATHF was further supported by the finding that maturation and the depletion of intracellular GTP by DDATHF were not reversed by guanine nucleosides in HL-60 cells deficient in hypoxanthine-guanine phosphoribosyltransferase activity. The findings provide support for the hypothesis that the terminal differentiation of these leukemic cells by DDATHF is the result of the depletion of intracellular GTP pools.

A study of toxicity and comparative therapeutic efficacy of vindesine-prednisone vs. vincristine-prednisone in children with acute lymphoblastic leukemia in relapse. A Pediatric Oncology Group study.

Vindesine (des-acetyl Vinblastine) is a synthetic derivative of vinblastine, and was produced with the hope that it would have less neurotoxicity and hematopoietic toxicity than other vinca alkaloids. Phase I and II studies also demonstrated significant activity in lymphoid malignancies, especially Acute Lymphoblastic Leukemia (ALL). The present study was designed to compare therapeutic effectiveness of twice weekly vindesine (2 mg/M2/dose) plus Prednisone (60 mg/M2/dose) (Treatment 1) to weekly Vincristine (2 mg/M2/dose) plus Prednisone (60 mg/M2/day) (Treatment 2). All patients were less than 21 years of age, and had documented bone marrow relapse (blast count > 25%). In 39 patients presumed sensitive to vincristine, there were 11 complete responses out of 20 patients (55%) randomized to receive vindesine/prednisone and 7 complete responses out of 19 patients (37%) treated with Vincristine/Prednisone. In 37 patients resistant to vincristine, there were 7 complete responses (19%). Vindesine was more toxic than Vincristine. Major toxicities of vindesine included paraesthesias, peripheral neuropathy and ileus. Vindesine hematological toxicity appeared greater, but such toxicity is hard to assess in patients with bone marrow disease. In this study, vindesine and vincristine had similar efficacy, but vindesine use was associated with more toxicity.